In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy

TY - JOUR

T1 - In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy

AU - Matic, Ivan

AU - van Hagen, Martijn

AU - Schimmel, Joost

AU - Macek, Boris

AU - Ogg, Stephen C.

AU - Tatham, Michael H.

AU - Hay, Ronald T.

AU - Lamond, Angus I.

AU - Mann, Matthias

AU - Vertegaal, Alfred C. O.

PY - 2008/1/1

Y1 - 2008/1/1

N2 - The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.

AB - The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.

KW - SUMO-2 TARGET PROTEINS

KW - QUANTITATIVE PROTEOMICS

KW - SUMOYLATION SITES

KW - CELL-CYCLE

KW - E3 LIGASE

KW - PATHWAY

KW - COMPLEX

KW - CHAINS

KW - YEAST

KW - UBC9

U2 - 10.1074/mcp.M700173-MCP200

DO - 10.1074/mcp.M700173-MCP200

M3 - Article

C2 - 17938407

SN - 1535-9476

VL - 7

SP - 132

EP - 144

JO - Molecular & Cellular Proteomics

JF - Molecular & Cellular Proteomics

IS - 1

ER -